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Research in Veterinary Science 75 (2003) 89–101
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Trace element, toxin and drug elimination in hair with
particular reference to the horse
M. Dunnett *, P. Lees
Department of Veterinary Basic Sciences, Royal Veterinary College, University of London, Hawkshead Campus, North Mymms, Hatfield,
Hertfordshire AL9 7TA, UK
1. Introduction
From a veterinary perspective, hair must be regarded
as a much neglected tissue. This is unfortunate, but not
surprising, since hair (as opposed to the skin from which
it grows) is not associated with major pathological disease and the physiology and biochemistry of hair in
animals have not been extensively studied. Whilst hair is
a living tissue, it is metabolically relatively inert and,
once formed, does not undergo further biogenic turnover. Its formation and growth is regulated by physiological processes, enabling responses, for example to
environmental influences. Whilst early anatomical
opinion viewed the skin and hair simply as a passive
barrier to fluid loss and mechanical injury, it is now
recognised that hair performs a range of integrated
functions (Table 1). For example, it is integral to body
temperature regulation and provides a protective barrier
against the animalÕs environment (Stenn and Paus, 2001;
Tregear, 1965). Thus, hair density is greater over regions
of the skin exposed to direct sunlight (Pilliner and Davies, 1996). Coat colour impacts on thermal regulation,
light coloured coats being more effective than darker
colours in hot environments (Lyne and Short, 1965;
Scott, 1988). In addition, glossiness of coat hair, as
found in tropical equine breeds, assists the reflection of
solar radiation (Hayman and Nay, 1961; Holmes, 1970).
In equine skin several hair types are recognised:
temporary hair makes up the majority of the coat; hair
of the mane, tail and eyelashes is permanent and tactile
hairs are located in or near the muzzle, ears and eyes.
The anatomical location of permanent hairs provides
protection in several ways. The mane assists the shedding of rainwater and insulates the head and major
*
Corresponding author.
E-mail address: [email protected] (M. Dunnett).
blood vessels to the brain (Pilliner and Davies, 1996),
whilst the eyelashes protect against corneal impact injury.
2. Hair structure, composition and growth
2.1. Hair shaft structure
The hair shaft derives from hair follicle growth.
Structurally, there are three distinct components: a central medulla, a protective outer cuticle and the cortex
located intermediately (Harkey, 1993). The ÔtiledÕ structure of the outer cuticle is due to overlapping cells, which
fix the hair shaft to the follicle by interlocking with cells
of the inner root sheath.
The greatest bulk of the hair shaft is made up of the
cortex containing longitudinally oriented, spindle
shaped keratinocytes. The cells are composed of macrofibrils, or keratin bundles, which comprise approximately 85% of the cortex (Cone and Joseph, 1996). The
keratin protein fibres cross-link to provide the hair with
its mechanical strength, and the structural proteins are
inter-spaced with air gaps termed fusi. The cortex also
contains melanin (eumelanin providing black/brown
pigmentation and pheomelanin red/yellow pigmentation) granules. Melanin is even more resistant than
keratin to enzyme and microbial attack. The medulla
comprises randomly orientated and loosely packed
rectangular cells, rich in the structural protein trichohyalin, which is less resistant than keratin. When dehydrated, medulla cells shrink to leave empty vacuoles
along the central axis of the hair shaft (Chatt and Katz,
1989). The numbers of medullary cells and hence medullary area increase with increasing hair fibre diameter.
Thus, the fine hairs of the equine coat are made up
mainly of cuticle and cortex cells, whilst mane and tail
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M. Dunnett, P. Lees / Research in Veterinary Science 75 (2003) 89–101
Table 1
Functions of hair (from Stenn and Paus, 2001)
Functions of hair
Insulation against heat loss and gain
Sensory assessment of the environment
Protection against trauma, insect penetration and electromagnetic radiation
Decoration, social communication and camouflage
Mechanism of outward transport of social environmental signals (sebum and pheromones)
hairs contain a greater number and a greater proportion
of medulla cells (Harkey, 1993; Talukdar et al., 1972).
Chemical composition of the shaft. Hair may be described as a crystalline, cross-linked and orientated
polymeric protein structure. As well as protein, the hair
shaft contains melanins, water, lipids and inorganic
minerals. Approximate percentage proportions in human
hair are, respectively, 80–85 (protein), 0.3–1.5 (melanins),
<15 (water), 1–9 (lipids) and 0.25–0.95 (minerals).
The principal hair proteins are three structurally related keratins, the low-sulphur, high-sulphur and hightyrosine, high-glycine keratins. The sulphur content
derives from sulphur-containing amino acids, principally cysteine. Hair lipids include free fatty acids and
triglycerides. The melanins are polymers formed in
melanocytes by the oxidation of tyrosine. The melanin
content of human hair has been found to vary between
individuals and between races (Borges et al., 2001). The
keratinised region of the hair shaft, extending beyond
the skin epidermis as visible hair, is dehydrated. Hair
water content derives from sweat and atmospheric
moisture; and varies directly with environmental humidity (Robbins, 1979). Several heavy metals, such as
lead, cadmium and mercury, and trace elements are
present in hair. The concentrations of these different
constituents vary with factors such as diet, disease, genetics and weathering.
Hair follicle structure. The hair follicle that forms the
hair shaft contains vascular, muscular and glandular
components (Chatt and Katz, 1989). Follicles vary in
structure with differing anatomical locations and are
thus capable of generating hair shafts of differing size,
shape, curl and colour (Stenn and Paus, 2001). Some
species, including dogs and cats, have compound hair
follicles which produce primary and secondary hairs,
whereas the horse has simple follicles that form exclusively single hairs (Lloyd, 1993; Talukdar et al., 1972).
Linked to each simple follicle are apocrine sweat and
sebaceous glands and an arrector pili muscle, contraction of which erects the hair shaft, thus regulating ventilation and heat loss. Erection is also associated with
the sympathoadrenal fight and flight response to perceived danger. Sebum is a lipid-based waxy substance,
formed by the sebaceous glands, which coats the hair
and skin to repel water and provide a physical barrier.
Sebum also inhibits the growth of microorganisms
(Lewis, 1995) and retards the penetration of toxic substances (Vale and Wagoner, 1997). The apocrine glands
excrete an oil that, like sebum, coats the hair.
The follicle also contains a dermal papilla, an inner
and outer root sheath, and a bulge region. The papilla
regulates follicular development by providing a permissive signal for hair growth. The hair bulb comprises
proliferative epithelial cells. These produce the hair
matrix and the inner and outer root sheaths (Lloyd,
1993). The hair follicle is an organ, containing several
enzyme systems, that determine the biochemical composition of hair (Jarrett, 1977; Potsch et al., 1997).
2.2. Hair growth
The rate of growth of human scalp hair is 0.7–1.5 cm/
month (Harkey, 1993). The hair shaft grows through the
formation of matrix cells within the bulb. Cell differentiation enables formation of the layers of the shaft and
the surrounding root sheaths. As the shaft reaches the
follicular bulge area, keratinisation leading to hardening
occurs by a process of protein cross-linking through
highly stable disulphide bridges between adjacent cysteine molecules. The relative resistance of hair to degradation is due largely to the cysteine cross-links. The
hair shaft then extrudes from the skin and the rate of
hair growth is determined by the rate of cell proliferation (Blume et al., 1991).
The growth cycle of hair follicles includes a long period of active hair growth (anagen), a short transitional
period of slow growth (catagen) and a rest period of no
growth (telogen), after which shedding of the hair shaft
(exogen) occurs (Harkey, 1993; Lloyd, 1993; Stenn and
Paus, 2001). In anagen the follicle actively forms new
hair shaft. In catagen new growth ceases and shrinking
of the follicle occurs until, in telogen, an inactive club
hair is formed (Randall and Ebling, 1991). As telogen
ends, a further anagen phase, involving regeneration of
hair matrix from stem cells in the permanent part of the
follicle, occurs under the regulatory control of the dermal papilla (Gailbraith, 1998). This leads to formation
of a new hair shaft and its growth causes shedding of the
previous club hair. At any one time, in human adults
approximately 85% of scalp hair is in the growing phase.
Although extensive data are not available, it is clear
that the length of the hair growth cycle and the duration
M. Dunnett, P. Lees / Research in Veterinary Science 75 (2003) 89–101
of each phase differ between individuals, species and
anatomical sites. The growth cycle provides the means
by which animals alter their pelage to meet the needs of
regeneration and seasonal fluctuations in climate (Randall and Ebling, 1991).
2.3. Non-dietary factors affecting hair growth rate in
horses
There is continual growth of the permanent hairs of
the equine mane and tail. Investigations in a small
numbers of horses over short intervals indicated a relatively constant rate of growth of mane hairs (Popot
et al., 2000; Whittem et al., 1998). Studies in our laboratory involving 29 horses of various breeds have shown
that both mane and tail hair growth is relatively constant over a 12-month period (Fig. 1). Month by month
comparisons indicated some variation in rates of hair
growth in both mane and tail, but overall there was no
clear correlation between these fluctuations and either
climatic or seasonal factors. Growth rate differed
slightly within three regions of the mane (Fig. 1), being
slowest near the withers, highest near the poll and intermediate near the crest. In both mane and tail, growth
rates were faster in native pony breeds than in thoroughbreds and intermediate in cross-breeds (Tracey
et al., 2002). This study indicated no demonstrable effect
of age or gender on rate of hair growth rate in the tail
and mane.
Seasonal hair growth and shedding of the pelage is
well recognised in horses and domestic pets, as well as
wild animals. Thus, in cattle and in cats hair growth is
absent or minimal in winter (Baker, 1974; Dowling and
Nay, 1960; Ryder, 1976). In sheep, wool growth rate
peaks in summer and early autumn (Coop, 1953), whilst
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in humans slightly faster hair growth occurs in late
summer and early autumn (Courtois et al., 1996; Randall and Ebling, 1991).
In spring, late summer and early autumn changes in
photoperiod, through the eyes and by several endocrine
pathways, affect hair growth. In the horse the onset of
pelage and the rate of shedding is increased by artificially extended photoperiods in both fillies (Wesson and
Ginther, 1982) and mares (Kooistra and Ginther, 1975;
Oxender et al., 1977). Pelage responses to photoperiod
change in pony colts were delayed, lagging behind day
length changes by 5–8 weeks (Fuller et al., 2001). Seasonally, the fastest growth occurs in the autumn (Popot
et al., 2000). The influence of photoperiod on the permanent hairs of the tail and mane has not been determined, but data from our laboratory suggests a
tendency for the growth of mane and tail hair to be
greatest in autumn.
Regulation of secretion of the pineal hormone melatonin in relation to mammalian pelage is mediated
through light receptors in the eye, which signal changes
in daylight length to the pineal gland. Melatonin synthesis and release increase as daylight length decreases
(Bergfelt, 2000).
The effect of androgenic steroids on hair growth in
horses is unknown, but red deer stags produce long
mane hairs in the breeding season under the influence of
androgens (Thornton et al., 2001). Circulating androgen
levels in humans may influence hair growth, although
this is not clearly established (Messenger, 1993; Randall
and Ebling, 1991).
In male horses, seasonal increases in blood prolactin
levels correlate with shedding of the winter coat (Argo
et al., 2001), and recombinant porcine prolactin
administration to seasonally anoestrous mares led to
Fig. 1. Mean cumulative mane and tail hair growth for a group of continuously grazes native ponies ðn ¼ 5Þ. Regions of the mane and tail were
shaved to the skin and subsequent re-growth was measured monthly over the subsequent 12 months.
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M. Dunnett, P. Lees / Research in Veterinary Science 75 (2003) 89–101
pelage shedding within 14 days (Thompson et al., 1997).
The thyroid gland also affects hair growth; enhanced
growth occurs in response to increased circulating thyroxine levels in human subjects (Parker, 1981) and dogs
(Gunaratnam, 1986). On the other hand, hypothyroidism is commonly associated with diffuse alopecia
(Ebling, 1981). Although data in horses are limited,
coarser coat hair growth has been reported in thyroidectomised mares (Lowe et al., 1987).
Whilst the effect of daylight length on melatonin and
prolactin secretion, and hence on pelage growth, is well
known, there is no information on the effects of these
hormones on equine mane and tail hair growth rates.
Likewise, there are few data on the influence of climatic
factors, such as temperature, intensity of solar radiation
and relative humidity. However, young standard-bred
horses when cold-housed produced up to twice as much
coat hair as warm-housed horses of the same age and
breed (Cymbaluk, 1990).
3. History of hair analysis
Casper (1857–1858) detected arsenic by hair analysis
11 years post-mortem in a suspected murder victim.
There seems to have been no further interest in hair
analysis for more than 80 years thereafter, until Flesch
(1945) suggested that hair might be regarded both as a
metabolic end product and excretory organ, the trace
element content of which reflected the medium from
which it was formed. Subsequently, the heavy metal
content of hair was described by Goldblum et al. (1953),
and the same group (Goldblum et al., 1954) provided
the first report of detection of an organic drug, phenobarbitone, in guinea pig hair. Both Forshufvud et al.
(1961) and Smith et al. (1962) undertook retrospective
hair analysis to investigate the possibility that the Emperor Napoleon had been poisoned with arsenic. Their
analysis revealed repeated exposure to arsenic. However, no firm conclusion regarding the arsenic source
could be drawn, since pigments containing arsenic were
used in wallpaper manufacture in the early years of the
19th century. Metabolites of cocaine and its parent
compound were detected retrospectively in Peruvian
mummy hair more than 500 years old (Springfield et al.,
1993).
Analysis of human hair to monitor nutritional trace
element content and to identify and track exposure to
heavy metals was used throughout the 1960s and 1970s.
An example is suspected mercury poisoning in Iraq,
resulting from consumption of bread prepared from
grain contaminated with mercury-based fungicides
(Giovanoli-Jakubczak and Berg, 1974). Likewise, hair
analysis was used to monitor occupational and lifestyle
exposures to such heavy metal toxins as mercury in
dental technicians (Leniham et al., 1973) and lead de-
riving from traffic exhaust emissions in school children
(Hammer et al., 1971).
Hair analysis as a means of detecting the abuse of
controlled drugs and establishing in individual human
subjects a history of drug use was introduced by
Baumgartner et al. (1979). This group detected opioid
drugs in hair samples from addicts by a radioimmunological method. Shortly thereafter, hair analysis was
extended to tracking other drugs of abuse, including
barbiturates (Smith and Pomposini, 1981), phencyclidine (Baumgartner et al., 1981) and cocaine (Valente
et al., 1981). Since then there have been major advances
in analytical methodology, sensitivity and validation to
enable detection of a wide range of drugs of abuse and
therapeutic agents in human hair (Gaillard and Pepin,
1999; Nakahara, 1999; Tagliaro et al., 1997).
Hair is not generally regarded as a major excretory
organ for endogenous or exogenous (including drugs
and toxins) compounds. Quantitatively, amounts eliminated in hair, expressed as a percentage of administered
dose, are inevitably small. However, compared to most
body tissues, hair (itself very resistant to environmental
forces) provides a very stable medium, in which trace
elements, minerals, drugs, toxins and their metabolites
can be protected and detected over prolonged periods.
Hair analysis can thus provide a historical record of
drug (or other chemical) exposure, even though some
losses may occur due to chemical change or leaching
out. As well as detecting drugs or chemicals retrospectively months or years after systemic exposure, hair root
analysis may indicate acute exposure. Gygi et al. (1995)
detected codeine in root hair one hour after administration. The attraction of hair as a matrix for analysis
lies also in the fact that it is easily collected, transported
and stored and methods for the extraction and chemical
analysis of a wide range of compounds at low concentrations have been established and validated. Potential
routes for the incorporation of drugs and other substances, including trace minerals, into hair are illustrated
in Fig. 2. Entry may be gained through capillaries supplying nutrients to the follicles, in sebum, oil or in sweat.
However, whilst the latter secretion is available to the
horse, sweating does not occur in the cat or dog.
4. Assessment of nutritional status by hair analysis
The assessment of nutritional status, including essential elements and trace minerals, by hair analysis has
been used for many years and within the last 20 years
increasing use has been made of spectroscopic methods
to facilitate multi-element analysis. Current analytical
techniques provide reliable, rapid and relatively inexpensive diagnostic methods (Chyla and Zyrnicki, 2000).
Hair contains high concentrations of many trace elements, and has been used to monitor nutritional status
M. Dunnett, P. Lees / Research in Veterinary Science 75 (2003) 89–101
Fig. 2. Proposed multi-compartment model for the incorporation of
drugs and other substances into hair (adapted from Henderson, 1993).
over extended time periods. Compared to other matrices, such as plasma and urine, hair analysis circumvents
the transient fluctuations arising from recent or variable
dietary intake.
Attempts have been made to use hair analysis as an
indicator of the whole body status of minerals, such as
calcium and phosphorus (Sippel et al., 1964; Wysocki
and Klett, 1971) and trace metals such as copper, molybdenum, zinc, selenium and iron (Cape and Hintz,
1982; Wichert et al., 2002). However, there are still uncertainties as to whether hair content is well correlated
with whole body levels and the validity of the approach
in the horse remains to be confirmed (Hintz, 2000).
5. Hair analysis to monitor environmental toxin and heavy
metal exposure
Hair analysis has been used to track the history of
human exposure to toxic heavy metals, such as cadmium, mercury and arsenic (Chatt and Katz, 1989).
There has been less extensive use of hair analysis in
animal toxicological studies. However, there are reports
of environmental exposure of wildlife to heavy metals
(Burger et al., 1994) and selenium (Clark et al., 1989;
Edwards et al., 1989), and to selenosis in domesticated
species (Mihajlovic, 1992). Lead levels in coat hair of
animals grazing pasture near to a lead smelter were
significantly increased (Levine et al., 1976). In horses in
central Europe, environmental exposure to cadmium in
relation to age, breed, gender and location has been
investigated by hair analysis. Cadmium accumulated to
a greater extent in geldings than mares (Anke et al.,
1989). The exposure of horses, sheep and alpacas to
several toxic heavy metals and other elements, including
cadmium, lead, chromium, nickel and bromine, from
vehicle emissions was investigated by hair analysis by
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Ward and Savage (1994). Increased lead and cadmium
concentrations were detected in equine hair and blood,
with a significant correlation between blood and hair
levels of lead. Toxic levels of selenium in forage were
strongly correlated with selenium concentrations in
coat, mane and tail hair. Hair selenium concentrations
ranged from 0.3 to 7.1 mg/kg (Witte et al., 1993).
In humans, pesticides including 1,1-dichloro-2,2-bis
(p-chlorophenyl)ethylene (DDE) and other polychlorinated biphenyls were detected in hair in concentrations
of 0.5–4.9 pg/kg (Dauberschmidt and Wennig, 1998).
The application of hair analysis to the detection and
monitoring of plant-induced toxicoses in horses would
be a useful application. It has been estimated that approximately 500 horses die each year from hepatic disease caused by the ingestion of Common (or Tansy)
Ragwort in the UK alone. The hepatotoxins causing
primary hepatic failure are pyrrolizidine alkaloids (Lewis, 1995). It is predictable that these and other alkaloids will be deposited in equine hair.
6. Drug elimination in equine and canine hair
6.1. Potential uses of drug detection and quantification in
hair
The elimination of drugs and their metabolites in hair
is potentially of great value in relation to sports antidoping control, as well as in pre-purchase vetting and
residue monitoring in stock production. Illegal drug use
in animals may arise in several ways. The growth-promoting properties of anabolic steroids, such as testosterone, nandrolone and stanozolol, and repartitioning
agents, including clenbuterol, albuterol and brombuterol, can be used illegally to enhance muscular development in equine bloodstock and cattle breeding
programmes. Thus, Appelgren et al. (1996) detected
clenbuterol in calf hair. In addition, a wide range of
performance altering drugs, including local anaesthetics,
CNS depressants and stimulants and drugs acting on
the cardiovascular system, may be misused both during training and prior to participation in competitive
equine and canine sports. Furthermore, anti-inflammatory corticosteroids such as dexamethasone and nonsteroidal anti-inflammatory drugs (NSAIDs), such as
phenylbutazone, may be used abusively prior to competition or to mask lameness in horses before prepurchase veterinary examination. A further potential
application of hair analysis is in the detection of drugs
used in therapy but which are not authorised for use in
horses intended for human consumption.
Recent equine studies have demonstrated the potential of hair analysis to provide additional analytical
evidence to that obtained from blood or urine analyses.
In contrast to urine and blood analyses, however, hair
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M. Dunnett, P. Lees / Research in Veterinary Science 75 (2003) 89–101
analysis can detect and quantify drugs weeks, months or
even years after administration or intake. A further
consideration is that racehorses may fail post-race tests
for prohibited substances through ingestion of a number
of contaminants naturally present in feedstuffs (Table 2).
In human medicine hair analysis has been used to
monitor compliance with long term therapeutic drug
regimens, for example for anti-epileptic drugs and
Dunnett et al. (2002a) have demonstrated a similar potential for phenobarbitone use in epileptic dogs.
6.2. Mechanisms and correlations of drug elimination in
hair
The elimination of drugs, and indeed other substances, in hair is not necessarily a passive process. The
supply, accumulation and subsequent persistence of
drugs in the hair shaft are achieved through a range of
poorly understood mechanisms, involving not only
physico-chemical properties of drugs but also depending
on the animalÕs anatomy and physiology. It is therefore
of interest to explore these inter-relationships, so that
the science of hair analysis may be taken beyond their
detection and quantitation. Accordingly, in 1998 a research programme in our laboratory commenced with
the aim of exploring possible correlations between drug
concentration in mane and tail hair on the one hand and
a range of factors such as administered dose, drug
pharmacokinetics and the constituents of hair on the
other. Our objectives have been to (a) establish and
validate methods of extraction and chemical analysis of
therapeutic and illicit drugs and their metabolites in
equine mane and tail hair; (b) evaluate the potential of
hair analysis for the retrospective detection of drug use
and misuse in horses, including investigation of the relationship between concentration and hair growth rates;
(c) establish relationships between administered dose
and pharmacokinetic parameters for drugs in plasma
(such as AUC and Cmax) and the level of accumulation
and persistence in hair; (d) determine the importance of
hair-related factors such as melanin content and drug
concentration achieved in hair, including tracking drug
concentration–time relationships by segmental analysis;
and (e) determine the influence of drug-related factors
such as lipid solubility, acid–base properties and binding
to plasma protein and the level of accumulation.
Regarding objectives (a) and (b), we have established
analytical methods for and detecting in equine hair
many of the drugs listed in Table 3. Regarding objectives (c–e), to penetrate cell membranes the drug must
have some degree of lipid solubility. Most drugs are
either weak acids or weak bases and are partially ionised
at physiological pHs. The unionised fraction is generally
lipid soluble and will penetrate cell membranes readily.
However, pH may differ intra- and extra-cellularly and
weak acids are diffusion trapped in alkaline environTable 3
Drugs detected to date in equine hair
Drug detected
Metabolite detected
Trimethoprima; d
Sulphadiazinea;d
Sulphadimidinee
Metronidazolea;d
Procainei
Enrofloxacine
Etamiphyllinec
Pentoxyfillinec
Caffeinec
Xylazineb
Methocarbamolb
Morphineh
Diazepamf
Clenbuterolf
Stanozololg
Boldenoneg
Salicylatee
Griseofulvine
Omeprazolee
Carprofene
No
No
No
No
No
Ciprofloxacin and others
Desethyletamiphylline and others
Lysofylline and others
Theobromine and theophylline
No
No
No
No
No
No
No
No
No
5-Hydroxyomperazole
No
a
Dunnett and Lees (2000).
Dunnett et al. (2002c).
c
Dunnett et al. (2002b).
d
Dunnett et al. (2003).
e
Dunnett and Lees (unpublished data).
f
Popot et al. (2000).
g
Popot et al. (2002).
h
Whittem et al. (1998).
i
Dunnett and Lees (2002).
*
Identity not confirmed.
b
Table 2
Prohibited substances present in feedstuffs
Chemical classification
Pharmacological classification
Examples
Alkaloid
Alkaloid
Alkaloid
Alkaloid
Organic acid
Volatile oil
Muscarinic receptor antagonists
Narcotic-analgesics
Phosphodiesterase inhibitors
Hepatotoxins
Anti-inflammatory-analgesic
Respiratory stimulant
Miscellaneous
Atropine and hysocine
Morphine
Caffeine, theophylline and theombromine
Pyrrolizidines
Salicylate
Camphor and menthol
Borneol, bufotenine, dimethyl sulphoxide,
hordenine, lupanine, oryzanol and sparteine
M. Dunnett, P. Lees / Research in Veterinary Science 75 (2003) 89–101
ments and vice versa for weakly basic drugs (Henderson–Hasselbalch mechanism). The isoelectric pH of hair
is close to 6 (more acid than plasma) and this favours
the accumulation of weakly basic drugs in matrix cells
(Robbins, 1979). However, there is an additional factor
that favours even greater accumulation of basic drugs;
melanocytes have an intracellular pH in the range of 3–5
and this leads to diffusion trapping of basic drugs. If
incorporated during melanin granule formation, they
may become bound to melanin and this entrapment of
95
basic drugs maintains the diffusion gradient down which
further drug can migrate by passive diffusion (Potsch
et al., 1997). With its high melanin content, black hair
concentrates basic drugs more effectively than white hair
and brown hair is intermediate (Gaillard and Pepin,
1999). This is illustrated for the fluoroquinolone antimicrobial drug enrofloxacin and its metabolite ciprofloxacin in Fig. 3. Fig. 4 presents percentage binding
versus concentration data for in vitro binding to melanin of six drugs. It will be seen that for the weak organic
Fig. 3. In vivo uptake of the fluoroquinolone antibiotic enrofloxacin and its major metabolite ciprofloxacin in black and white equine mane and tail
hair. Hair was collected from a single bi-coloured horse one month after oral administration of the drug at 5 mg/kg body weight for 10 days.
Fig. 4. In vitro drug-melanin binding of six drugs.
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M. Dunnett, P. Lees / Research in Veterinary Science 75 (2003) 89–101
acids, phenylbutazone, sulphadiazine and phenobarbitone, binding is much lower than for the weak organic
bases, trimethoprim and procaine. These data also indicate that binding becomes saturable at high drug
concentrations, as reflected in a decrease in percentage
binding. Melanin binding (affinity) has also been proposed as a possible explanation for the long-term retention of isoxsuprine in the horse as evidenced by the
detection of this drug in post-race urine samples long
after treatment was reported to have ceased (Torneke
et al., 2000).
Nakahara (1999) proposed that the ratio, drug concentration in hair:plasma AUC, should be used as an
index of drug incorporation tendency and as a basis for
understanding incorporation mechanisms. For human
hair the highest ratio was obtained for cocaine and the
lowest was for 11-nor-tetrahydrocannabinol-9-carboxylic acid, the difference being 3600-fold. Current studies
in our laboratory have established correlations between
administered dose and hair concentration. This is illustrated for the organic bases procaine and trimethoprim
and the organic acid sulfadiazine in Fig. 5. With in-
Fig. 5. Dose vs. hair drug concentration relationships for three drugs. Hair samples were collected up to three months post-clinical treatment with
either intramuscular procaine benzylpenicillin or oral potentiated sulphonamides.
Fig. 6. Effect of incubation time on the extent of drug extraction. Samples of equine mane hair (approx. 20 mg) were incubated in 0.1 M hydrochloric
acid at 65 °C.
M. Dunnett, P. Lees / Research in Veterinary Science 75 (2003) 89–101
creasing dose the highest hair concentrations were obtained for trimethoprim and the lowest concentrations
for sulfadiazine.
6.3. Analytical methods
The principal analytical method of analysis used in
our laboratory has been that of high pressure liquid
chromatography, although for potent drugs, adminis-
97
tered in small amounts and present in hair in low concentrations, GC/MS methods may be required. Limited
penetration into hair may also be associated with rapid
clearance and short elimination half-life and with very
high levels of binding to plasma protein, for example
with NSAIDs.
For quantitative hair analysis, as well as requisite
levels of sensitivity, chemical methods must be validated
for accuracy and precision. Other important concerns
Fig. 7. Influence of successive washing procedures on extraction of procaine from equine hair.
Fig. 8. Influence of wash time on recovery (expressed logarithmically) of enrofloxacin from two sections of equine hair (0–50 and 200–250 mm from
the root) and of ciprofloxacin on one section of equine hair (0–50 mm from the root).
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M. Dunnett, P. Lees / Research in Veterinary Science 75 (2003) 89–101
are extraction and clean up/washing procedures. The
former must achieve a high and consistent percentage of
drug present, whilst the latter must not only remove
interfering substances but also allow distinction between
drugs present as true excreta after systemic administration or ingestion and drug present as contaminant introduced by contact with urine, faeces or other
environmental elements. The effect of incubation (extraction) time on amount of drug present in equine hair
for four drugs is illustrated in Fig. 6. It will be seen
that little further extraction is obtained when incubation exceeds 24 h. The influence of washing/extraction
procedure on drug content of hair and amounts removed by washing is illustrated in Figs. 7 and 8. Fig. 7
illustrates losses of procaine arising from, successively,
two buffer washes, two water washes and two acetone
washes. The total loss was 16.5%. Fig. 8 illustrates the
effect of wash time on the recovery of enrofloxacin and
ciprofloxacin from equine tail hair. It will be seen that
losses were small in relation to amounts of the fluoroquinolones extracted.
6.4. Summary of findings and future prospects
To date, studies in our laboratory have shown that
several anti-microbial drugs, including sulphonamides,
trimethoprim, metronidazole (Fig. 9 illustrates the extraction of metronidazole from an extract of mane hair),
enrofloxacin and procaine benzylpenicillin can be detected in mane and tail hair samples up to 2 years (and
in one instance 3 years) after systemic administration.
This implies virtually indefinite stability, although a
steady decrease over time was reported by Dunnett et al.
(2002a), after therapeutic administration of phenobar-
Fig. 9. Upper panel: Chromatogram showing the presence metronidazole in an extract of equine mane hair. Lower panel: Comparative UV spectra
derived from the metronidazole peak in the analysed hair sample and that from a drug standard (Dunnett et al. (2003), reproduced with permission).
M. Dunnett, P. Lees / Research in Veterinary Science 75 (2003) 89–101
bitone to dogs for epilepsy management. Declining drug
concentrations may be due to decomposition caused by
heat or UV light or hair damage leading to drug leakage
(Dunnett et al., 2002b; Dunnett and Lees, 2000). Further studies have also demonstrated the detection of
several methylxanthine drugs, including caffeine and
theobromine and their metabolites, in mane and tail hair
(Dunnett et al., 2002b). Other investigators have detected performance-modifying drugs, including morphine (Beresford et al., 1998), diazepam and clenbuterol
(Popot et al., 2000) in equine hair. However, cocaine
was not detected in mane hair following systemic administration (Whittem et al., 2000). Studies in progress
in our laboratory have extended drug analyses in hair
and correlations between administered dose and drug
pharmacokinetics to corticosteroids, NSAIDs and sedatives of various classes. It is clear that there are virtually no limitations to qualitative and quantitative
analyses of drugs and their metabolites in equine hair.
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